LAUSR

Nucleobase oxidation and alkylation can destroy Watson-Crick base-pairing to challenge the genomic integrity. Human 8-oxoguanine glycosylase 1 (hOGG1) and alkyladenine glycosylase (hAAG) are evolved to counter these two cytotoxic lesions through base-excision repair, and their deregulations are implicated with multifactorial diseases and cancers. Herein, we demonstrate activatable self-dissociation of Watson-Crick structures with fluorescent nucleotides for sensing multiple human glycosylases at single-cell level. The presence of hOGG1 and hAAG catalyzes 8-oxoG and deoxyinosine removal in functional probe 1 to release two trigger probes (1 and 2). Then, trigger probes hybridize with functional probe 2 to activate the autocatalytic degradation of functional probes 2 (Cycle I) and 3 (Cycle II), replicating abundant trigger probes (1-4) and releasing two fluorophores (2-aminopurine (2-AP) and pyrrolo-dC (P-dC)). New trigger probes (1, 2) and (3, 4), in turn, hybridize with free functional probes 2 and 3, repeating Cycles I and II turnovers. Through multicycle self-dissociation of Watson-Crick structures, 2-AP and P-dC are exponentially accumulated for the simultaneous quantification of hOGG1 and hAAG. This nanodevice exhibits high sensitivity with a detection limit of 2.9 × 10-3 U/mL for hOOG1 and 1.5 × 10-3 U/mL for hAAG, and it can measure enzymatic kinetics, identify potential inhibitors, discriminate glycosylases between cancer and normal cell lines, and even quantify glycosylase activities in a single HeLa cell. Moreover, this assay may be rapidly and isothermally performed in one tube with only one tool enzyme in a quencher-free manner, promising a simple and powerful platform for multiple human glycosylase detection.